Our specific aims are two, each scheduled for one year of study. First, we will determine if the regulatory and catalytic subunit of cAMP-dependent protein kinase is externally located on rat epididymal (caput vs. caudal) sperm. Second, we will determine if Ca++ concentrations affect the ability of cAMP to bind to the regulatory subunit and relatedly determine if Ca++ concentrations increase or decrease sperm protein phosphorylation caused by the catalytic subunit. Our attached preliminary data support both specific aims as being valid hypostheses to test. All methodology required is currently in use by the P.I and their use substantitated by his publications. Specific methods will include: (1) 125I-lactoperoxidase iodination and/or galactose oxidase catalyzed (3H)NaBH4 labeling of external membrane proteins with a double labeling protocol using a photoaffinity probe, (32P)-8N3-cAMP. The photoaffinity probe labels only the regulatory subunit of the enzyme; (2) using the photoaffinity probe of ATP, (32P)-8N3-ATP, we will determine he effects of various Ca++ concentrations on sperm proteins that bind ATP (in the presence of UV light) and compare these sperm proteins to those sperm proteins that are phosphorylated (in the absence of UV light or labeled only with 32P-ATP); and (3) general laboratory procedures of SDS-PAGE electrophoresis coupled with molecular weight determinations of the separated proteins, differential centrifugation, liquid scintillation counting, and autoradiography are required to obtain the above data. Our long term objectives focus on the importance of the two specific aims, which in turn are pertinent to the observations that caput sperm are immature and incapable of fertilization, while caudal sperm are mature (acquire motility) and carry out normal fertilization. Considerable data exists to support the hypothesis that this post-testicular sperm maturation may require a Ca++ -dependent cAMP-dependent protein kinase system. What is unique about our research is that we are proposing an external location for the sperm enzyme. Numerous studies, not yet possible, would be revealed that are important to the regulation of sperm function by genital track fluids if this external enzyme hypothesis is accepted.